Method and Apparatus for Delivery of Submicroliter Volumes onto a Substrate

ABSTRACT

A slotted pin tool, a delivery system containing the pin tool, a substrate for use in the system and methods using the pin tool and system are provided. The slotted pin tool contains a plurality of pins having slotted ends designed to fit around each loci of material deposited on a surface, such as a microarray, without contacting any of the deposited material. Sample is delivered by contacting the pin tool with the surface; the amount delivered is proportional to the velocity of the pin tool as it contacts the surface or the velocity of the liquid when movement of the pin is halted.

RELATED APPLICATIONS

Benefit of priority under 35 U.S.C. §119(e) to U.S. provisionalapplication Ser. No. 60/244,404, filed Oct. 30, 2000, to Chao Lin etal., entitled “METHOD AND APPARATUS FOR DELIVERY OF SUBMICROLITERVOLUMES ONTO A SUBSTRATE” is claimed herein. The subject matter of theprovisional application is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to sample dispensing systems and, moreparticularly, to the delivery of liquid samples onto substrate, such asa microarray, for laboratory analysis.

BACKGROUND Description of the Background Art

Genetic sequencing efforts, such as the Human Genome project, haveproduced vast amounts of information for basic genetic research thathave proven useful in developing advances in health care and drugresearch. These advances are possible because of improvements inengineering and instrumentation that provide advanced tools for thebiotechnology community to continue with basic genetic research. Withthese advances, scientists can move from basic genomic discoveries toassociating specific phenotypes and diseases, and can thereby betteridentify targets for drug development.

Nucleic acid sequencing and diagnostic methods often analyze samplesdeposited onto target locations on substrates microarrays, such hasmicroplates, silicon chips and other such supports capable of retainingbiological molecules or samples at discrete loci. Microarrays have beenused to execute tests on large batches of genetic samples to generatephenotype associations and improve interpretation of the large data setsthat result from such tests. A typical microarray, referred to as achip, includes a substrate, such as a silicon or silicon-coatedsubstrate, on which a large number of reactive points receive samplesfor testing. Microarray chips provide a technology that permitsoperators to increase sample throughput, allowing the screening of largenumbers of samples and reducing reagent costs by using submicrolitersample volumes. Preparation of such arrays employs a variety ofmethodologies, including printed arrays and spotted arrays, with a widevariety of substrate surfaces and different modes of quantification. Theresulting microarrays are used as substrates for a variety ofbiochemical applications.

Among the ways for delivery of multiple samples to loci on microarraysurfaces are solid pins. The solid pins typically are dipped into aliquid sample, which coats the tip of each pin, holding a sample dropletby surface tension. The coated pins are then touched to a target surfaceon a microarray substrate, so that the sample is transferred to thetarget by contact printing. The size and taper of the pin tool tip canaffect the volume of liquid sample that is picked up during dipping. Theamount of liquid sample transferred on contact will vary with thesurface tension of the liquid. Pin tools also can be problematic forhigh throughput systems because the pins may have to be changed ifdifferent sample volumes are desired, or if the nature of the liquidsample is changed to avoid sample contamination. In addition, pin toolscannot be used in situations where contact dispensing where there is arisk of damage to a fragile preloaded sample, such as for massspectrometric analyses in which samples are deposited on loci that havepreloaded material, such as matrix material for matrix-assisted laserdesorption (MALDI).

Some mass spectrometry formats, such as MALDI formats, combine thesample to be tested with a matrix material, such as an inorganic acid,which when dried forms a crystal structure. Matrix material can bepreloaded on a mass spectrometry substrate and the sample can be addedat a later time, using an appropriate liquid dispensing apparatus. Whena sample target is preloaded or prespotted with the porous matrixmaterial required for mass spectrometry, direct contact by the solid pinwith the matrix material can crush the material.

Other liquid samples dispensing apparatus rely on piezoelectricmechanisms, sometimes using quill-type pin tools that hold the samplesin a cut-out at the lower tool tip. Such piezoelectric delivery systemsare susceptible to dispensing satellite droplets on a target locationbecause of surface tension effects. Piezoelectric systems also may beprone to variations in voltage and frequency among different tips, whichresults in variation between the volume of liquid sample dispensed fromdifferent individual tips.

From the discussion above, it is apparent that there is a need for adispensing systems that can accurately deposit precise amounts of liquidsample on target locations on a substrate, with a high throughput rate,without risk of cross contamination of samples or damage to thedeposited material. Therefore, it is an object herein to provideapparatus, methods and substrates for fulfilling these and other needs.

SUMMARY OF THE INVENTION

A delivery system for delivery of precise amounts small volumes,particularly submicroliter and smaller volumes is provided. Alsoprovided are pin tools for use in the system and substrates forretaining samples, particularly substrates for use in the systemsprovided herein.

One delivery system with pin tool as constructed as provided herein,accurately delivers small volumes, typically submicroliter or nanoliteror picoliter volumes, of liquid samples onto a substrates, such as amicroarray substrate, at high throughput rates by dipping a slotted pintool (a pin tool having one or more pins with slotted ends) having anopen tip into a sample reservoir or well containing a liquid sample tobe delivered onto the substrate, thereby drawing a volume of liquidsample up into the pin tool. The slotted pin tool is then moved towardthe substrate at a predetermined rate and then is halted, therebyexpelling the liquid sample from the slotted pin tool onto the reactionlocation of the substrate. Thus, the sample fluid is expelled from theslotted pin tool by the force of momentum. The volume of liquid sampleexpelled is proportional to the momentum of the moving pin tool (i.e.,the amount delivered is proportional to the velocity of the pin tool asit contacts the surface or to the velocity of the liquid in a pin whenmovement of the pin tool is halted). Hence volume delivered is afunction of the speed of moving the pin tool toward the microarray,which provides a way to accurately control and deliver desired samplevolumes. For each pin tool size there is a range of volumes in which theamount of volume delivered is a linearly of the velocity of the pintool. Sample volume delivered is not dependent on tip surface areal,thereby providing for flexibility in use since it is not necessary tochange pins to dispense different volumes.

The system uses the slotted pin tool provided herein. The pin tool has aslot that is sufficiently large to contain the volume of sample liquiddesired for delivery. The slotted pin tool and system are providedherein. In one aspect, the pin tool slot may be sized to fit aroundtarget locations, such as loci on which material has been deposited on asubstrate, such as a microarray substrate, to prevent contact betweenthe pin tool and the material. The slotted pins can be mounted in aholding block so as to move up and down in the block; the positions ofeach pin in the block are selected to match the target loci on thesubstrate. The slotted pins in the pin tool have a substantiallycylindrical tip having a lateral slot forming a cavity with a width ofgreater than at least about 10, 30, 50, 75 or 100 μm, and can be of asize up to about 300 μm or 500 μm or 1000 μm and having a height of atleast about, 25, 50 μm, 100 μm or greater. The selected size is afunction of the delivered volume of liquid. Such pins can deliversamples of as low or lower about 1 nanoliter and higher, and can be aslow as about 3-10 picoliters.

For example, a pin tool provided herein with a 300 μm slot permitsdelivery of volumes of as low as about 1 nanoliter to 30 nanoliters. Forthis pin tool and for delivery of volumes in this range the volumedelivered is linearly related to the velocity of the tool prior tohalting it. Varying the size of the slot permits greater variation involume delivered.

The particular geometry of the slot in the pin tool is selected as afunction of the size of the loci on the target array. In someembodiments, system is designed so that the pin tool halts prior tocontacting the surface. In other embodiments it contacts the surface.For embodiments in which the halting of the movement of the pin toolresults from or includes contact with the substrate, the slots fitaround each locus.

Generally the sample selected to be delivered, when intended for massspectrometric analysis by MALDI, results in a spot on the substratesurface that is at least the size of the laser spot but can be smalleror larger as desired. A typical laser spot is about 30-50 μm. Deliveryof about 5 nanoliters results in a spot of about 100 μm. The precisesize of the spot varies depending upon the surface on which it isdelivered.

To move the pin tool towards the substrate, the holding block can bemoved toward the substrate, such as a microarray substrate, until theslotted pins on the tool make contact with the microarray, whereupon thepin tool tips fit around the loci, such as spots of matrix material,without contacting any deposited material on the surface. The pin toolthen moves upward in the pin tool holding block, which is then movedaway from the microarray. Because it is designed to fit around eachlocus, the pin tool does not contact any material, such as matrixmaterial for MALDI, cells, protein crystals or other materials, on thesubstrate. In this way, the dispensing system accurately depositsprecise amounts of liquid sample on target locations, such as on amicroarray substrate, with a high throughput rate, without contacting ordamaging any material, such as matrix material, deposited on asubstrate.

A microarray substrate that can be used with the system is alsoprovided. This microarray is constructed using photolithographictechniques and hydrophobic materials. Target locations on the microarrayare defined with the application of photoresist materials andphotolithographic deposition to create an array of locations on the chipthat are less hydrophobic than the surrounding areas. The differentialhydrophobicity confines the droplets to a desired locus. The microarrayscan contain any desired number of loci from 1 to 1000, to 2000 or more,and typically have 96-, 384-, 1536-loci. Higher densities are alsocontemplated. The pins in the pin tools are in a pattern that matches aselected array.

By virtue of the pin tool design herein, it is possible to transfer thesample to a pre-determined locus on a substrate that already haspre-deposited material, such as matrix, cells, such as bacterial ormammalian cells, protein crystals and other materials sensitive tocontact. Since the instant tools provided herein rely on inertial forcesfor delivery, delivery of liquids is primarily dependent upon themomentum of the liquid in the slotted tool, not on the relative surfacetensions of the pin and the substrate for the liquid. As one result, thepin tools provided herein permit accurate and controlled delivery ofdefined volumes by selection of the velocity of the tool at impact or asit reaches it the substrate and is stopped prior to contact.

Substrates that contain two materials, a photoresist material treated torender it resistant to chemical treatments such as silation used massspectrometry and other synthetic procedures, and a second morehydrophobic material are provided. Unlike most substrates that employphotolithographic methods, the photoresist is not removed from thesurface, but includes the target loci of the surface. This is achievedby baking the substrate. Hence a substrate that contains photoresistmaterial as the target loci are provided.

Also provided are combinations of pin tools that contain slotted pinsand substrates, where the number and arrangement of pins and size of theslots is designed to match the arrayed loci and, preferably, the slotsare of a size that is greater than each locus, or each locus with loadedor preloaded material, such as matrix material.

Other features and advantages of the apparatus and methods providedherein should be apparent from the following description of preferredembodiments, which illustrate, by way of example, the principles of themethods and apparatus and substrates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a sample delivery system constructed as provided herein.

FIG. 2 is a perspective view of a pin tool block and its use in thedelivery system shown in FIG. 1.

FIG. 3 is a block diagram illustrating the primary components of adelivery system shown in FIG. 1.

FIG. 4 is a side phantom view of a slotted pin tool in the sampledelivery system illustrated in FIG. 1, illustrating a “floating pin”configuration.

FIG. 5 is a detail side view of a slotted pin tool of the FIG. 1 system.

FIG. 6 is a plan view of the slotted pin tool of FIG. 5, looking downthrough the pin tool toward the microarray substrate.

FIGS. 7A, 7B, and 7C are side views of the slotted pin tool showing theliquid sample as drawn into the pin tool and deposited onto thesubstrate.

FIG. 8 is a side view of an alternative embodiment of a slotted pin toolfor the FIG. 1 system, illustrating a spring-loaded configuration.

FIGS. 9A, 9B, and 9C are side views of an alternative embodiment of apin tool for the FIG. 1 system, illustrating a solenoid-activated hollowpin tool.

FIG. 10 is a plan view of a microarray substrate for use in the FIG. 1sample delivery system.

FIG. 11 is a side view of an alternative embodiment of a slotted pintool having a tapered end permitting entry into very small wells.

FIG. 12 is an enlarged view of the tip of the pin tool illustrated inFIG. 11.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. In the event there are differentdefinitions for terms herein, the definitions in this section control.Where permitted, all patents, applications, published applications andother publications and sequences from GenBank and other data basesreferred to throughout in the disclosure herein are incorporated byreference in their entirety.

Among the issued patents and published international applicationsincorporated by reference are: U.S. Pat. Nos. 5,807,522, 6,110,426,6,024,925, 6,133,436, 5,900,481, 6,043,031, 5,605,798, 5,691,141,5,547,835, 5,872,003, 5,851,765, 5,622,824, 6,074,823, 6,022,688,6,111,251, 5,777,324, 5,928,906, 6,225,450, 6,146,854, 6,207,370,International PCT application Nos. WO 99/12040, WO 97/42348, WO98/20020, WO 98/20019, WO 99/57318, WO 00/56446 and WO 00/60361. Thesepatents and publications describe a variety of mass spectrometricanalytical methods, substrates and matrices used in mass spectrometricanalyses, and related methods and apparatus, including pin tools andother dispensing systems. It is intended that the methods, substrates,pin tools and delivery systems provided herein are for use in place oraddition to the delivery methods, apparatus and substrates described andused in these patents and patent applications. Other intended usesinclude any methods and assays that use microarrays and other suchsubstrates for syntheses and screening, including sequencing,oligonucleotide and protein syntheses and diagnostic assays, and areparticularly suitable for use in high throughput formats.

As used herein, a molecule refers to any molecule or compound that islinked to a substrate. Typically such molecules are macromolecules orcomponents or precursors thereof, such as peptides, proteins, smallorganics, oligonucleotides or monomeric units of the peptides, organics,nucleic acids and other macromolecules. A monomeric unit refers to oneof the constituents from which the resulting compound is built. Thus,monomeric units include, nucleotides, amino acids, and pharmacophoresfrom which small organic molecules are synthesized.

As used herein, macromolecule refers to any molecule having a molecularweight from the hundreds up to the millions. Macromolecules includepeptides, proteins, nucleotides, nucleic acids, and other such moleculesthat are generally synthesized by biological organisms, but can beprepared synthetically or using recombinant molecular biology methods.

As used herein, the term “biopolymer” is used to mean a biologicalmolecule, including macromolecules, composed of two or more monomericsubunits, or derivatives thereof, which are linked by a bond or amacromolecule. A biopolymer can be, for example, a polynucleotide, apolypeptide, a carbohydrate, or a lipid, or derivatives or combinationsthereof, for example, a nucleic acid molecule containing a peptidenucleic acid portion or a glycoprotein, respectively. The methods andsystems herein, though described with reference to biopolymers, can beadapted for use with other synthetic schemes and assays, such as organicsyntheses of pharmaceuticals, or inorganics and any other reaction orassay performed on a solid support or in a well in nanoliter or smallervolumes.

As used herein, a biological particle refers to a virus, such as a viralvector or viral capsid with or without packaged nucleic acid, phage,including a phage vector or phage capsid, with or without encapsulatednucleotide acid, a single cell, including eukaryotic and prokaryoticcells or fragments thereof, a liposome or micellar agent or otherpackaging particle, and other such biological materials. For purposesherein, biological particles include molecules that are not typicallyconsidered macromolecules because they are not generally synthesized,but are derived from cells and viruses.

As used herein, the term “nucleic acid” refers to single-stranded and/ordouble-stranded polynucleotides such as deoxyribonucleic acid (DNA), andribonucleic acid (RNA) as well as analogs or derivatives of either RNAor DNA. Also included in the term “nucleic acid” are analogs of nucleicacids such as peptide nucleic acid (PNA), phosphorothioate DNA, andother such analogs and derivatives or combinations thereof.

As used herein, the term “polynucleotide” refers to an oligomer orpolymer containing at least two linked nucleotides or nucleotidederivatives, including a deoxyribonucleic acid (DNA), a ribonucleic acid(RNA), and a DNA or RNA derivative containing, for example, a nucleotideanalog or a “backbone” bond other than a phosphodiester bond, forexample, a phosphotriester bond, a phosphoramidate bond, aphosphorothioate bond, a thioester bond, or a peptide bond (peptidenucleic acid). The term “oligonucleotide” also is used hereinessentially synonymously with “polynucleotide,” although those in theart recognize that oligonucleotides, for example, PCR primers, generallyare less than about fifty to one hundred nucleotides in length.

Nucleotide analogs contained in a polynucleotide can be, for example,mass modified nucleotides, which allows for mass differentiation ofpolynucleotides; nucleotides containing a detectable label such as afluorescent, radioactive, luminescent or chemiluminescent label, whichallows for detection of a polynucleotide; or nucleotides containing areactive group such as biotin or a thiol group, which facilitatesimmobilization of a polynucleotide to a solid support. A polynucleotidealso can contain one or more backbone bonds that are selectivelycleavable, for example, chemically, enzymatically or photolytically. Forexample, a polynucleotide can include one or more deoxyribonucleotides,followed by one or more ribonucleotides, which can be followed by one ormore deoxyribonucleotides, such a sequence being cleavable at theribonucleotide sequence by base hydrolysis. A polynucleotide also cancontain one or more bonds that are relatively resistant to cleavage, forexample, a chimeric oligonucleotide primer, which can includenucleotides linked by peptide nucleic acid bonds and at least onenucleotide at the 3′ end, which is linked by a phosphodiester bond, orthe like, and is capable of being extended by a polymerase. Peptidenucleic acid sequences can be prepared using well known methods (see,for example, Weiler et al., Nucleic acids Res. 25:2792-2799 (1997)).

A polynucleotide can be a portion of a larger nucleic acid molecule, forexample, a portion of a gene, which can contain a polymorphic region, ora portion of an extragenic region of a chromosome, for example, aportion of a region of nucleotide repeats such as a short tandem repeat(STR) locus, a variable number of tandem repeats (VNTR) locus, amicrosatellite locus or a minisatellite locus. A polynucleotide also canbe single stranded or double stranded, including, for example, a DNA-RNAhybrid, or can be triple stranded or four stranded. Where thepolynucleotide is double stranded DNA, it can be in an A, B, L or Zconfiguration, and a single polynucleotide can contain combinations ofsuch configurations.

As used herein, the term “polypeptide,” means at least two amino acids,or amino acid derivatives, including mass modified amino acids and aminoacid analogs, that are linked by a peptide bond, which can be a modifiedpeptide bond. A polypeptide can be translated from a polynucleotide,which can include at least a portion of a coding sequence, or a portionof a nucleotide sequence that is not naturally translated due, forexample, to its location in a reading frame other than a coding frame,or its location in an intron sequence, a 3′ or 5′ untranslated sequence,a regulatory sequence such as a promoter. A polypeptide also can bechemically synthesized and can be modified by chemical or enzymaticmethods following translation or chemical synthesis. The terms“polypeptide,” “peptide” and “protein” are used essentially synonymouslyherein, although the skilled artisan recognizes that peptides generallycontain fewer than about fifty to one hundred amino acid residues, andthat proteins often are obtained from a natural source and can contain,for example, post-translational modifications. A polypeptide can bepost-translationally modified by, for example, phosphorylation(phosphoproteins), glycosylation (glycoproteins, proteoglycans), whichcan be performed in a cell or in a reaction in vitro.

As used herein, the term “conjugated” refers stable attachment,typically by virtue of a chemical interaction, including ionic and/orcovalent attachment. Among preferred conjugation means are:streptavidin—or avidin—to biotin interaction; hydrophobic interaction;magnetic interaction (e.g., using functionalized magnetic beads, such asDYNABEADS, which are streptavidin-coated magnetic beads sold by Dynal,Inc. Great Neck, N.Y. and Oslo Norway); polar interactions, such as“wetting” associations between two polar surfaces or betweenoligo/polyethylene glycol; formation of a covalent bond, such as anamide bond, disulfide bond, thioether bond, or via crosslinking agents;and via an acid-labile or photocleavable linker.

As used herein, “sample” refers to a composition containing a materialto be detected. In a preferred embodiment, the sample is a “biologicalsample” (i.e., any material obtained from a living source (e.g. human,animal, plant, bacteria, fungi, protist, virus). The biological samplecan be in any form, including solid materials (e.g. tissue, cell pelletsand biopsies) and biological fluids (e.g. urine, blood, saliva, amnioticfluid and mouth wash (containing buccal cells)). Preferably solidmaterials are mixed with a fluid. In particular, herein, the samplerefers to a mixture of matrix used or mass spectrometric analyses andbiological material such as nucleic acids. The pin tools and systemsprovided herein are designed to dispense nucleic acid compositions intomatrix that has been deposited on a substrate or to dispensecompositions containing matrix material and biological material such asnucleic acids onto a selected locus or plurality of loci on a substrate.

As used herein, a composition refers to any mixture. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a combination refers to any association between amongtwo or more items. The combination can be two or more separate items,such as two compositions or two collections, can be a mixture thereof,such as a single mixture of the two or more items, or any variationthereof.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, the term “solid support” means a non-gaseous, non-liquidmaterial having a surface. Thus, a solid support can be a flat surfaceconstructed, for example, of glass, silicon, metal, plastic or acomposite; or can be in the form of a bead such as a silica gel, acontrolled pore glass, a magnetic or cellulose bead; or can be a pin,including an array of pins suitable for combinatorial synthesis oranalysis.

As used herein, “substrate” refers to an insoluble support onto which asample and/or matrix is deposited. Support can be fabricated fromvirtually any insoluble or solid material. For example, silica gel,glass (e.g. controlled-pore glass (CPG)), nylon, Wang resin, Merrifieldresin, Sephadex, Sepharose, cellulose, magnetic beads, Dynabeads, ametal surface (e.g. steel, gold, silver, aluminum, silicon and copper),a plastic material (e.g., polyethylene, polypropylene, polyamide,polyester, polyvinylidenedifluoride (PVDF)). Exemplary substrateinclude, but are not limited to, beads (e.g., silica gel, controlledpore glass, magnetic, Sephadex/Sepharose, cellulose), capillaries, flatsupports such as glass fiber filters, glass surfaces, metal surfaces(steel, gold, silver, aluminum, copper and silicon), plastic materialsincluding multiwell plates or membranes (e.g., of polyethylene,polypropylene, polyamide, polyvinylidenedifluoride), pins (e.g., arraysof pins suitable for combinatorial synthesis or analysis or beads inpits of flat surfaces such as wafers (e.g., silicon wafers) with orwithout plates. The solid support is in any desired form, including, butnot limited to: a bead, capillary, plate, membrane, wafer, comb, pin, awafer with pits, an array of pits or nanoliter wells and othergeometries and forms known to those of skill in the art. Preferredsupport are flat surfaces designed to receive or link samples atdiscrete loci. Most preferred as flat surfaces with hydrophobic regionssurrounding hydrophilic loci for receiving, containing or binding asample.

As used herein, the term “target site” refers to a specific locus on asolid support upon which material, such as matrix material, matrixmaterial with sample, and sample, can be deposited and retained. A solidsupport contains one or more target sites, which can be arrangedrandomly or in ordered array or other pattern. When used for massspectrometric analyses, such as MALDI analyses, a target site or theresulting site with deposited material, is preferably equal to or lessthan the size of the laser spot that will be focussed on the substrateto effect desorption. Thus, a target site can be, for example, a well orpit, a pin or bead, or a physical barrier that is positioned on asurface of the solid support, or combinations thereof such as a beads ona chip, chips in wells, or the like. A target site can be physicallyplaced onto the support, can be etched on a surface of the support, canbe a “tower” that remains following etching around a locus, or can bedefined by physico-chemical parameters such as relative hydrophilicity,hydrophobicity, or any other surface chemistry that retains a liquidtherein or thereon. A solid support can have a single target site, orcan contain a number of target sites, which can be the same ordifferent, and where the solid support contains more than one targetsite, the target sites can be arranged in any pattern, including, forexample, an array, in which the location of each target site is defined.The pin tools provided herein contain blocks that hold the pins in apattern that matches the pattern of target sites on a the support, suchthat upon contacting the support, the ends of the pins surround, but donot touch each loci nor any of the loci.

As used herein, the term “predetermined volume” is used to mean anydesired volume of a liquid. For example, where it is desirable toperform a reaction in a 5 microliter volume, 5 microliters is thepredetermined volume. Similarly, where it is desired to deposit 200nanoliters at a target site, 200 nanoliters is the predetermined volume.

As used herein, the term “liquid dispensing system” means a device thatcan transfer a predetermined amount of liquid to a target site. Theamount of liquid dispensed and the rate at which the liquid dispensingsystem dispenses the liquid to a target site.

As used herein, the term “liquid” is used broadly to mean a non-solid,non-gaseous material, which can be homogeneous or heterogeneous, and cancontain one or more solid or gaseous materials dissolved or suspendedtherein.

As used herein, the term “reaction mixture” refers to any solution inwhich a chemical, physical or biological change is effected. In general,a change to a molecule is effected, although changes to cells also arecontemplated. A reaction mixture can contain a solvent, which provides,in part, appropriate conditions for the change to be effected, and asubstrate, upon which the change is effected. A reaction mixture alsocan contain various reagents, including buffers, salts, and metalcofactors, and can contain reagents specific to a reaction, for example,enzymes, nucleoside triphosphates, amino acids, and the like. Forconvenience, reference is made herein generally to a “component” of areaction, wherein the component can be a cell or molecule present in areaction mixture, including, for example, a biopolymer or a productthereof.

As used herein, submicroliter volume, refers to a volume convenientlymeasured in nanoliters or smaller and encompasses, for example, about500 nanoliters or less, or 50 nanoliters or less or 10 nanoliters orless, or can be measured in picoliters, for example, about 500picoliters or less or about 50 picoliters or less. For convenience ofdiscussion, the term “submicroliter” is used herein to refer to areaction volume less than about one microliter, although it will bereadily apparent to those in the art that the systems and methodsdisclosed herein are applicable to subnanoliter reaction volumes aswell.

As used herein, nanoliter volumes generally refer to volumes betweenabout 1 nanoliter up to less than about 100, generally about 50 or 10nanoliters.

As used herein, with respect to the supports provided herein, an elementis defined as less hydrophobic than another by the relative“wettability” of the element or contact angles, where the contact angleof an element is less than the surrounding surface. The contact angle isthe angle the breaks the surface tension when a liquid is delivered. Ahydrophilic substrate requires a relatively lower contact angle than amore hydrophobic material. Hence contact angle refers to relativehydrophobicity between or among surfaces.

As used herein, high-throughput screening (HTS) refers to processes thattest a large number of samples, such as samples of diverse chemicalstructures against disease targets to identify “hits” (see, e.g., Broachet al. High throughput screening for drug discovery, Nature, 384:14-16(1996); Janzen, et al. High throughput screening as a discovery tool inthe pharmaceutical industry, Lab Robotics Automation: 8261-265 (1996);Fernandes, P. B., Letter from the society president, J. Biomol.Screening, 2:1 (1997); Burbaum, et al., New technologies forhigh-throughput screening, Curr. Opin. Chem. Biol., 1:72-78 (1997)]. HTSoperations are highly automated and computerized to handle samplepreparation, assay procedures and the subsequent processing of largevolumes of data.

As used herein, a photoresist refers to a photoresist obtained bypolymerization of a diazo photosensitive material with a phenol resin.These photoresists are generally called positive type photoresists.

As used herein, symbology refers to a code, such as a bar code or othersymbol, that is engraved, stamped or imprinted on a substrate. Thesymbology is any code known or designed by the user. In general, thesymbols are identifiable to the user or are associated with informationstored in a computer or memory and associated with identifyinginformation.

As used herein, the abbreviations for amino acids and protective groupsand other abbreviations are in accord with their common usage and, ifappropriate, the IUPAC-IUB Commission on Biochemical Nomenclature (see,(1972) Biochem. 11: 942-944).

Delivery System

As noted the delivery system provided herein delivers small volumes,typically submicroliter volumes, of liquid samples onto a substrate athigh throughput rates by dipping a slotted pin tool having an open tipinto a sample reservoir or well containing a liquid sample to bedelivered onto a substrate, thereby drawing a volume of liquid sample upinto the pins in the pin tool. The pin tool with slotted pin(s) is movedfrom the sample well to an elevated position above a reaction locationon the microarray that is to receive the liquid sample, is loweredtoward the substrate at a predetermined speed, and then the movement ofthe pin tool toward the substrate is halted, thereby expelling theliquid sample from the slotted pin tool onto the reaction location ofthe substrate, such that the sample fluid is expelled from the slottedpin tool by the force of momentum. The volume of liquid sample expelledis determined by the speed of moving the pin tool toward the microarray.Typically the pin tool contains a plurality of slotted pins. In certainembodiments, the outer surface of the pin is rendered hydrophobic, suchas by silanation or other chemical means, relative to the inner surfaceto thereby reduce or eliminate any satellite drops that adhere to theouter surface.

FIG. 1 depicts an exemplary instrument for pin tool based dispensing.Any such instrument may be adapted for use with the pin tool providedherein. FIG. 1 shows a sample delivery system 100 constructed asdescribed herein. The system 100 includes a base table 102 on which ismounted a transport system 104 having rails or runners that can move asample in x, y, and z planar coordinates relative to the table and tomicrotiter plates containing reagents. The x-direction (which will alsoreferred to as left-right) is indicated in FIG. 1 by the arrows 106, they-direction (also referred to as top-bottom) is indicated by the arrows108, and the z-direction (also referred to as up-down) is indicated bythe arrows 110.

A pin tool holding block 112 is mounted to the transport system 104 formovement in the y-direction along a y-axis rail 114 and for movement inthe z-axis direction along a z-axis rail 116. Microtiter plates 118mounted on an MTP table 120 are moved in the x-axis direction along anMTP rail 122, to move back and forth relative to the pin tool holdingblock 112. The target microarray chips 124 are mounted on a target table126 and are moved in the x-axis direction along a target rail 128 fordelivery of liquid samples, as described further below.

The pin tool holding block 112 is illustrated in FIG. 2, which showsthat the holding block holds a plurality of pin tools 202. The number ofpin tools held in the holding block will typically correspond to thenumber of target locations on the microarray chip 124 (FIG. 1) that willreceive samples. In genomic research, for example, a microarraysubstrate chip may typically contain ninety-six or more targetlocations. Microarray chips with other numbers of target locations mayalso be used, such as 384-target arrays or even 1536-target arrays.

The pin tool holding block 112 may also include a single-pin holdingstation 204, where a single pin tool may be attached. The single holdingstation permits one pin tool to be easily attached for single-pointprocessing or other special operations involving a single pin tool. Thesingle pin holding station 204 is preferably located on the block 112such that the single pin tool may be easily accessed for attachment andremoval from the pin tool block 112 during normal system operation.

As described further below, the sample delivery system 100 can deliveraccurately-controlled volumes of liquid samples onto target locations ofa microarray substrate at high throughput rates by dipping a slotted pintool into a reservoir of liquid sample, thereby drawing a volume ofliquid sample up into the pin tool, then moving the slotted pin tool toa position above the substrate, lowering the slotted pin tool toward thesubstrate at a predetermined speed, and then abruptly halting themovement of the pin tool and expelling the liquid sample from the pintool onto the substrate. The sample is expelled due to the momentum ofthe liquid, which is still traveling at the speed of the pin tool whenthe pin tool is halted. Thus, using the momentum delivery techniquedescribed herein, the liquid sample can be deposited onto the microarraysubstrate without making extended contact with the substrate and withoutcontact between the pin tools and any material at a locus or loci on thesubstrate.

As described further below, the movement of a pin tool toward themicroarray 124 may be halted either because the slotted pin tool makescontact with the microarray, or because the pin tool reaches the limitof travel relative to the pin tool holding block 112. If the pin toolmakes contact with the microarray during downward movement of theholding block, then the pin tool is preferably mounted in the block soas to move upwardly independent of the block, so that the pin tool canmove up while the block itself is moving downward. In such anarrangement, the pin tool contacts the microarray and the block isstopped in its downward movement substantially simultaneously, followedby lifting up the holding block, carrying the pin tools with it.Alternatively, the pin tool may be moved independently from the block112 toward the microarray by an actuating mechanism such as a solenoid,halting when the pin tool reaches the limit of travel in the actuatingmechanism. These alternatives are described further below.

It has been found that the speed of lowering the pin tool preciselydetermines the volume of the liquid sample that is expelled. That is,the slot of the pin tool is loaded with liquid sample by dipping, andthe portion of that loaded sample that will be expelled is determined bythe speed of lowering the pin tool. The momentum delivery technique ofthe provided herein utilizes any pin tool lowering speed that willimpart a momentum force to the liquid sample that is greater than thesurface tension of the liquid. The delivery system 100 permits precisecontrol over the speed of moving the pin tool toward the microarraysubstrate 124. The delivery system thereby carefully controls deliveryof the liquid sample, and expels the samples from the pin tools withreduced contamination problems and with increased efficiency andthroughput.

Process Steps for Sample Delivery

To deliver the samples to the microarray 124, the pin tool holding block112 is moved across the table 102 and the pin tools are moved verticallyin the z-direction 110 as described above. The pin tool holding block112 is mounted to an arm 130 of the transport system 104. As the arm 130moves along the y and z rails 114, 116, the holding block 112 is movedas well, and can thereby be moved across the stations of the table 102for sample delivery onto microarray substrate chips.

The pin tools are moved first to an ultrasonic cleaning station 132 thatincludes a cleaning solution bath 134 into which the tips of the pintools are dipped. The ultrasonic cleaning will typically requireapproximately five to ten seconds to sufficiently clean the pin tooltips of any remaining samples from prior system operation. When a pintool is being used on an initial operation, a longer cleaning time isusually necessary, to remove contaminants from the manufacturingprocess. Such initial cleanings are referred to as preconditioning, andtypically require approximately thirty seconds in the ultrasonic bath134.

The next station of the system 100 is a rinsing and drying station 136.This station includes an empty recess into which the pin tools arelowered, whereupon the recess is filled with a rinse solution, such asdistilled water. The rinse bath maintains the pin tool tips in asubmerged state for a duration of one to ten seconds, as required forsufficient cleaning of the sample fluid being processed. At theconclusion of the submerged time, the rinse solution is drained, and anair bath is begun, for drying the pin tools. In the preferredembodiment, a vacuum is used in the bottom of the recess to draw off anyexcess rinse solution and empty the recess. The vacuum drying time istypically on the order of one to ten seconds duration.

Next, the pin tool holding block 112 is moved to a microtiter plate thatincludes wells containing the liquid sample to be delivered. The holdingblock is moved from the rinsing and drying station 136 to a positionover the MTP table 120. The MTP table may include multiple microtiterplates; only one such microtiter plate 118 is shown in FIG. 1 forsimplicity of illustration. Each microtiter plate will preferablycontain as many sample wells as there are target locations on thedestination microarray 124. The illustrated system 100 has a capacity often microtiter plates, as well as a capacity of ten microarray chips,but it should be apparent that different capacities can be easilyprovided, as desired. A system operator can specify the location of thesource microtiter plate 118 in x-y coordinates of the MTP table 120, andalso can specify the x-y location of the destination chip 124, with auser control interface described further below, or automatic modes ofoperation can be implemented to move the pin tools from the variouspreparatory stations 132, 134 and from one microtiter to the next forcontinuous processing, as desired. The MTP table 120 will move along theMTP rail 122 and the holding block 112 will move along the y-rail 114 incooperation to position the desired microtiter plate beneath the holdingblock.

When the pin tool holding block 112 is located over the appropriatemicrotiter plate on the MTP table 120, the holding block will be loweredso that the pin tool tips are dipped into the liquid sample contained inthe microtiter wells. The volume of liquid sample that will be drawninto the slots of the pin tools will be determined by the size of theslots and the surface tension of the liquid sample. The duration orholding time of the pin tools in the microtiter wells, as well as thespeed of lowering, can be selected by the system operator. The durationand speed of dipping will be selected by the operator for the desiredsample volume, in accordance with the nature of the sample and factorssuch as sample viscosity, temperature, and the like.

After the liquid sample has been drawn into each pin tool slot and thepin tool holding block 112 has been raised up from the MTP table 120,the holding block will be moved over an appropriate microarray chip 124for sample delivery. The chip is located along the microarray chip rail128, on which multiple chips may be located, and will be moved intoproper position for receiving liquid samples. Only one microarray chip124 is shown in FIG. 1 for simplicity of illustration, but the preferredembodiment permits as many chips to be located on the chip rail 128 asthere are microtiter plates 118 on the MTP table 120.

The proper alignment of the pin tools over target locations of theappropriate chip is a critical process, and can be accomplished forexample, by the system 100 with a robotic vision unit 140. Initialalignment for a particular pin tool can be accomplished in a number ofways, For example, a camera is mounted on the machine that seeks thetarget. To align the pin tool with the target loci it is necessary tolocate pin(s) relative to the target and/or the pins. To locate thepins, the pins are, for example, dipped into a dye or ink and thencontacted with a blank substrate. The camera and software therefor the“learns” or images the locations of the spots and can then direct thepin tool to the corresponding positions on the actual substrate.Alternatively, other marks can be used. Transparent sticky tape can beplaced on the surface of the blank substrate and the pin touched thereonto imprint its image on the tape. The camera with software can thenlearn the locations of the pins. This procedure can also be automated.Such procedure should be performed for each pin tool to create an imagethereof so that the loci on the substrate and the pins can be properlyaligned.

The robotic vision unit includes a camera 142 that is mounted above thepin tool holding block 112, having a field of view that encompasses atleast one corner of the microarray chip that is beneath the holdingblock. The image that should be observed in the camera field of viewwhen the holding block is properly positioned is known (i.e., see, forexample, above). Therefore, the system 100 can confirm properpositioning by comparing the image being received from the camera 142with the known image that should be obtained.

The system 100, for example, can check for the appearance of a knownregistration mark that is imprinted on the microarray chip 124, toconfirm that the mark is in the expected location, or the system cancheck for the presence of a target location on the chip that should bein a known position in the camera field of view when there is properregistration of the pin tool block. If the expected registration mark ortarget location does not appear in the expected position, then thesystem will issue a warning to the operator and will halt sampleprocessing.

The system may perform a pattern recognition operation to check forproper positioning of the microarray chip and also check for proper chipcomposition. The image that is obtained in the camera field of view canbe compared with the image that should be obtained with a properlyproduced and aligned chip. Any anomalies in the obtained image mayindicate a defective chip, or may indicate that the chip is misaligned.In either case, a warning may be provided and operation of the systemmay be automatically halted. After the situation with the defective orimproperly positioned chip has been corrected, the system operator canindicate to the system that it should continue with normal processing.

If the pin tool holding block 112 is properly positioned, as confirmedby the robotic vision unit 140, then the pin tools are moved toward themicroarray chip 124 at a predetermined speed for a time sufficient toimpart downward momentum to the liquid sample volumes contained withinthe slots of the pin tools. The pin tools are then halted in theirtravel and, because the liquid samples still have downward momentum, theliquid samples keep moving at their imparted speed and therefore areexpelled from the pin tools. The liquid samples therefore aresimultaneously deposited on the target locations of the chip 124.

After the samples have been deposited on the microarray, the pin toolholding block 112 is moved from its position over the chip 124 and ismoved back to the starting point for operations, which is the cleaningstation 132. Alternatively, the holding block may be moved to a homeposition, as commanded by the system operator. The process of cleaning,rinsing and drying, dipping, and expelling may then be repeated, withcorresponding movement of the microtiter plates 118 and microarray chips124 for dipping and expelling, respectively.

System Control

FIG. 3 is a schematic block diagram illustrating the primary componentsof the delivery system shown in FIG. 1. A controller 302 contains acomputer processor and associated software and circuitry to communicatewith and control the mechanisms of the sample table 304. The sampletable 304 as illustrated in FIG. 3 represents the table mechanisms shownin FIG. 1 for moving the pin tools along their respective rails, and formoving the microarray chips along the microtiter rail. The table 304also includes the mechanisms for controlling the operation of thecleaning station, rinsing and drying station, dipping station, androbotic vision unit described above.

The controller 302 is itself controlled by a user interface device 306,such as a conventional laptop or desktop Personal Computer withapplication software to provide a graphical user interface. The systemoperator may adjust the speed of downward pin tool movement and mayspecify target microarray chips and microtiter plates, as well as otheroperational parameters, through the user interface device 306.

Pin Tools

The pin tools provided herein are include pins that are slotted suchthat upon contacting the surface of a substrate with deposited materialor other loci, the pin tool contacts the surface of the substrate, butdoes not touch any of the loci or material deposited thereon. Theslotted pins are also provided, as is a block containing or othersupport containing a plurality of pins in an array or arrangement thatmatches the array of loci on a target surface.

The individual pins in the pin tools 202 (FIG. 2) are of slottedconstruction to work with the momentum delivery technique of the system100. A pin tool 202 is shown in side section in FIG. 4, illustrating a“floating” pin arrangement. A detail side view of a pin tool tip isshown in FIG. 5 and a plan view through the pin tool tip is shown inFIG. 6.

FIG. 4 shows a pin tool 202 in the pin tool holding block 112 andindicates that the holding block is hollow, having a top wall 402 andbottom wall 404 joined by a side wall 406. It should be understood thatonly one pin tool is shown for simplicity of illustration, but that theholding block has a greater holding capacity, as described above. Thepin tool 202 slides up and down relative to the holding block 112through an upper guide hole 408 in the top wall 402 and a lower guidehole 410 in the bottom wall 404. A clip 412 attached to the pin toolprevents the pin tool from falling out through the bottom wall. Thus,the pin tool is free to move upwardly in the holding block 112. When theholding block is moved toward the microarray 124, the pin tool is freeto make contact with the surface of the microarray without sufferingdamage and without damaging any material on the microarray, and theholding block 112 can be set to move upward at that point, through theuser interface. By permitting upward movement of the pin tool 202relative to the holding block 112, there is a greater operating marginfor setting the point at which the holding block will be moved backupward away from the microarray. This reduces the risk of damage.Moreover, this construction permits the pin tool to be abruptly haltedin downward movement (by making contact with the microarray), therebyexpelling the liquid sample, without damaging the pin tools ormicroarray.

When a pin tool 202 makes contact with the microarray, no materialdeposited on the microarray will be damaged, because the tip of the pintool has a slotted construction. FIG. 4 shows a slot 420 in the tip ofthe pin tool. The slot is sized to fit around material deposited at atarget location on the microarray.

FIG. 5 shows side detail of a slotted pin tool that is constructed asdescribed herein, and FIG. 6 is a plan view. FIG. 5 and FIG. 6illustrate how the pin tool preferably fits around the depositedmaterial. In FIG. 5, the pin tool 202 is shown with the slot 420 havingsufficient width to permit a mound of material 502 on a microarray 124to fit within the slot. Other than the slot 420, the pin tool 202 is asolid core construction. FIG. 6 is a plan view, showing that the slot420 is cut through the pin tool 202 so that the material 502 fits withinthe slot. Alternatively, the pin tool may be constructed with a hollowcore.

For a conventional microarray, the matrix material spots on themicroarray surface are typically greater than 100 μm in diameter, andfrequently approximately 200 μm in diameter. The distance from thecenter of one target location (or spot with deposited material, such asmatrix for MALDI) to the center of an adjacent target location (ormatrix material spot) on the microarray is typically approximately 4.5mm. Other target spacing may be used as well, including more densespacing of 2.25 mm from center to center or less dense spacing of 9 mm.Accordingly, the slot 420 preferably has a width of approximately 300μm, which is a width that provides a sufficient margin of error inpositioning of the pin tool and production of the material so that thepin tool safely fits around the typical material spot. The outerdiameter of the pin tool is typically approximately 600 μm.

The height of the slot 420 is approximately 5 mm, a height that resultsin a desired volume of liquid sample being drawn into the slot bycapillary action when the pin tool is immersed in the well of amicrotiter plate 124 at the MTP table 120 of the system. The systemoperator will adjust the dipping control mechanism, in concert with theexpected depth of reagent in the microtiter wells, through the userinterface so that the pin tool is lowered into the microtiter well justbelow the height of the slot 420 and is raised out of the well before abubble of air can form in the top of the slot and become trapped. If abubble is trapped, the volume of liquid sample drawn into the slot maybe imprecise, and the volume that is expelled may likewise be imprecise.Thus, FIG. 5 and FIG. 6 show a slotted pin tool 202 that has a slotvolume for containing liquid sample of approximate dimensions 300 μm×600μm×5 mm. The volume of liquid sample drawn into the slot typicallycontains a volume of between 50 nl and 100 μl. Those skilled in the artwill appreciate that this volume represents a larger dispensing volumethan obtainable with conventional pin tool delivery systems, thuseliminating evaporation problems that might otherwise arise with smallersample volumes.

FIGS. 7A, 7B, and 7C are side views of the slotted pin tool 202 showingan operational sequence of the liquid sample after it is drawn into thepin tool and then as it is deposited onto the microarray 124. Only onepin tool is illustrated, but it should be understood that the sequencedepicted applies to all the pin tools mounted in the pin tool holdingblock 112. In FIG. 7A, the pin tool has been dipped into the microtiterwell and liquid sample has been drawn into the slot, and the holdingblock 112 is moving toward the microarray 124.

In FIG. 7B, the pin tool holding block 112 has been lowered sufficientlysuch that the slotted end of the pin tool has made contact with themicroarray surface, fitting around the material as described above.Thus, the downward movement of the pin tool (depicted in FIG. 7A) hasbeen abruptly halted in FIG. 7B. The pin tool floats in the holdingblock, and therefore the position of the pin tool in FIG. 7B relative tothe holding block is somewhat elevated. Due to the momentum of theliquid sample, the liquid continues in the downward direction when thepin tool is halted, expelling the liquid sample out of the pin tool slotand onto the microarray. At approximately the same time when the pintool makes contact with the microarray, the holding block is movedupward and away from the microarray, thereby eliminating any significantdelay in processing. Moving the pin tool block without significant delayincreases the system throughput and maintains efficiency. FIG. 7C showsthe holding block 112 lifted upward, away from the microarray 124, withthe liquid sample remaining behind on the microarray.

FIG. 8 is a side view of an alternative embodiment of a pin toolmounting block 800 and pin tool 802 for the FIG. 1 system, illustratinga spring-loaded configuration. As with the FIG. 4 configuration, the pintool holding block 800 has a hollow construction, with an upper wall 804and a bottom wall 806 through which the pin tool 802 may slide up anddown. In FIG. 8, however, a spring 808 is attached around the pin tool,between the upper wall and bottom wall. A spring clip 810 fixes thebottom of the spring to a point on the shaft of the pin tool. When thepin tool makes contact with the surface of the microarray 124, the pintool will begin to move upward in the holding block 800. Because thespring 808 is fixed to the pin tool, the top of the spring is compressedagainst the upper wall 804, thereby cushioning the upward movement ofthe pin tool. The spring constant of the spring 808 may be selected forthe desired operation and action.

FIG. 8 also shows that a slotted pin tool constructed as describedherein may have a tip having a taper, rather than the cylindricalconstruction of the FIG. 4 embodiment. The FIG. 8 pin tool embodiment,for example, has an outside diameter at the tip of approximately 600 μm,and has an outside diameter at the full extent of the pin tool shaft ofapproximately 1.5 mm. The greater shaft diameter away from the pin tooltip provides a more durable construction and easier handling, while thenarrower tip diameter permits dispensing of small, microliter samplevolumes and also denser packing of the pin tool tips. As with the FIG. 4embodiment, the volume of liquid sample carried by the pin tool 802 willbe determined by the volume of the pin tool slot 812. In both the FIG. 4and FIG. 8 embodiments, the pin tool slot preferably has a width that istypically greater than 200 μm, to fit around material on the microarray,and has a slot height of approximately 100 μm (0.1 mm) to 5 mm,depending on the sample volume desired. The desired volume of liquidsample to be drawn in for delivery will typically be between 50 nl and100 μl.

In any of the pin tool embodiments described herein, the interior of thepin tool slot may be coated with an ion exchange resin or other resin toassist with cleaning the slot or condition the liquid sample prior todispensing. The increased size of the pin tool slot illustrated abovecompared with conventional quill-type pin tools permits reducedmanufacturing costs and easier slot inspection for signs of wear. Thepin tools provided herein are configured, through controlled downwardspeed and through spring loading (FIG. 8), such that the pin tools makecontact with the microarray surface at a carefully controlled speed,thereby reducing the amount of wear typically experienced by other pintool configurations.

FIGS. 9A, 9B, and 9C are side views of an alternative embodiment of apin tool for the FIG. 1 system, illustrating a solenoid-activated hollowpin tool. A metal pin tool 902 or array of pin tools are held in a pintool holding block 904 by metal e-clips 906. The movement of the pintool is limited to a vertical direction. The holding block itself can bemounted to any x-y-z station. As shown in FIGS. 9A, 9B, and 9C, the pintool is spring loaded. A spring 908 having a spring constant “D” isconnected to the pin tool and to the holding block, so that the springcan be loaded by pulling the pin tool in the vertical (z) direction.Because both ends of the spring are connected to the holding block andto the pin tool, the pin tool reaches a potential energy E_(pot(spring))defined by:

E _(pot(spring))=½Ds ²,

for a spring constant “D” when shortened by the distance “s”. The pintool itself does not contain a slot, such as illustrated in FIG. 4, butrather has a hollowed opening at its lower tip, similar to the end of acapillary tube. A volume of liquid sample is aspirated into the pin toolusing capillary action, when the pin tool is dipped into a microtiterplate or other sample well. The size of the hollowed opening defines theamount of liquid sample that will eventually be dispensed. The volume ofsample that fills the hollowed opening of the pin tool by capillaryaction will be the same as the dispensed volume. This provides animportant means of controlling the volume of liquid sample that isdispensed.

Samples are dispensed by loading the spring 908 and bringing the springto the potential energy level indicated by the equation above. Thespring is loaded (FIG. 9B) by mechanical compression or byelectromagnetic force such as supplied by a solenoid. In FIG. 9A, FIG.9B, and FIG. 9C, a solenoid 910 is shown at the top end of the pin tool,but other configurations for loading the spring 908 will occur to thoseskilled in the art. When the spring is released, the pin tool is movedtoward the microarray 124 at a predetermined velocity, given by theequation above, carrying the liquid sample in the hollowed opening andimparting it with the same velocity as the pin tool. When the pin toolreaches the end of its travel, at FIG. 9C, the pin tool stops in theholding block, but the liquid sample continues moving, due to theimparted momentum. Therefore, the liquid sample is expelled from thehollowed opening and is delivered to the microarray via momentum force.

FIG. 11 is a side view of an alternative embodiment of a slotted pintool 1100 constructed as described herein, having a tapered end. Asillustrated in FIG. 11, the upper portion 1102 of the pin tool towardthe holding block has a nominal outer diameter of approximately 1.6 mmand a length of 63.5 mm. The outer diameter of the pin tool shaft beginsto taper from the nominal diameter at a location 1104 approximately 12mm from the tip of the pin tool. The dimensions of the tapered end,described further below, have been found to provide accurate dispensingof nanoliter and subnanoliter volumes. The taper of the pin tool permitsdipping the pin tool into relatively small volume wells withoutcontacting the sidewalls of the wells, thereby permitting small volumesof liquid to be drawn into the pin tool. The taper of the pin tool alsodecreases the importance of pin tool alignment with the wells, as thereis increased clearance between the pint tool and the sidewalls of thewells. The decreased time used for pin tool alignment increases theefficiency of the dispensing system.

FIG. 12 shows an enlarged view of the slotted pin tool 1100 illustratedin FIG. 11, showing details of the pin tool tip. The upper locationwhere the taper begins is again indicated by the arrow 1104,approximately 12.0 mm above the pin tool tip. The slot of the pin toolextends from the tip to a location 1120 that may be from approximately0.1 mm to 5.0 mm above the tip, depending on the sample volume that isto be dispensed. The greater the height of the slot, the greater thevolume of sample liquid that can be drawn into the pin tool anddispensed therefrom.

At the pin tool tip 1122, the outer diameter of the pin tool isapproximately 0.4 mm to 0.6 mm. The width of the slot is preferablyapproximately 0.3 mm. These dimensions permit convenient use with samplewells of decreased diameter, and provides increased tolerances formisalignment. The taper from the upper taper location 1104 to the tip1122 is generally a linear taper, from about 1.6 mm diameter to about0.6-0.4 mm diameter, such that half of the taper diameter occursapproximately half way between the upper taper point 1104 and the lowertaper point 1122. It should be understood, however, that differentdimensions and taper configurations may be employed, and are a functionof various parameters, including the configuration of sample wells beingused and sample volumes that are desired.

Substrates

Any substrate suitable for biological and chemical reactions and assays,such as diagnostic and hybridization assays in which samples aredeposited at discrete loci is contemplated for use herein. The loci onthe substrates and pin tools are matched so that the pattern of pins andsize of the slots matches the arrangement and size of loci withpreloaded material thereon. Preferably, the number of pins is the sameas the number of loci or the loci are a multiple thereof to permitdeposition of material at a plurality of loci. Combinations of thesubstrates and the pin tools are also provided.

Substrates with microarrays in which a relatively hydrophilic region orcontact region is surrounded by a more hydrophobic area and methods forpreparation of such substrates are provided herein. The substratesurface is any surface that has an available reactive group, such as —OHor a primary amine, or is derivatized to have such group. Surfacesinclude but not limited to TEFLON® (polytetrafluoroethylene (PFTE);Trademark, E. I. DuPont), glass, derivatized glass, plastics, silicon,silicon dioxide (SiO₂) and any other such materials known to those ofskill in the art.

Also provided are methods of producing substrates and the resultingsubstrates that have contact angles that result in hydrophobic focusingof hydrophilic liquids on loci formed from photoresist materials. Theresulting substrates include elements (loci) on a surface that are lesshydrophobic than the surrounding surface, where hydrophobicity ismeasured by the relative wettability (relative contact angel) of thesurrounding area compared to each locus (element). The contact angel ofeach element is less than that of the surrounding surface. To producesuch arrays, a surface, such as any of those described herein or knownto those of skill in the art to be suitable for linking or retainingmacromolecules, including biopolymers, such as silicon or SiO₂ is coatedwith photoresist, covered with a mask that blocks light as loci on thesurface, and exposed to light, the photoresist in the unmasked portionsis washed off. The resulting surface is baked to render the photoresiststable to chemical treatments such as silation. The surface is thensilated. Since the silane does not stick to photoresist, the resultingsurface has silated regions that surround the photoresist elements atthe loci. Examples 1 and 3 exemplify this process and the resultingsubstrates with patterned microarrays. The substrates are preferablyabout 3000 mm×2000 mm, such as 3068 mm×1960 mm, or can be smaller orlarger. The number of elements (loci) on each substrate can be anydesired number, such as, 8, 16, 24, 96, 384, 1536, higher densities orany convenient number. Other combinations of surface materials in whichthe contact angel between the two surfaces is less than or equal to 20°C. are contemplated.

The step of baking the photoresist on the target loci is renders thesurface resistant to chemical treatments, such as silation. Theselection of the temperature and time is selected so that thephotoresist does not become too hydrophobic relative to the rest of thesurface for liquid to be focussed at the target loci. Baking should beperformed at temperatures of about 190-200° C. for at least about 50-70minutes. The temperature and time are variable and can be empiricallydetermined for the particular photoresist materials and time of bakingto obtain the requisite chemical resistance and stability to treatmentand hydrophobicity. Both parameters are important for production ofsurface with the requisite properties so that the surface can be treatedand used in analyses, such as mass spectrometry, and the two materialshave the appropriate relative hydrophobicity/hydrophilicity to achievehydrophobic focussing of the droplets on the target loci.

Photoresist Materials for Preparation of the Substrates

Many photoresist materials are known to those of skill in the art andare readily available. For use herein, selection among such material ismade and the materials are tested. Select from among the available thosethat when spun or coated on a surface and baked as described herein havea contact angle of no greater than 70° C. where the surrounding area isabout 90° C., or that have a relative contact angle that is less thanthe surrounding surface that results in a hydrophobic/hydrophilicfocussing of sample material on the loci. For example, a differential ofat least about 20° C. is suitable for use in substrates intended formass spectrometric analysis. The differential is such that it provides awettable surface.

The photoresist is from the class of commercially available diazoquinonecontaining positive photoresists (see U.S. Pat. Nos. 3,402,044,2,797,213, 3,148,983, 3,046,118, 3,201,239, 3,046,120, 3,184,310,3,567,453, 4,550,069, 5,607,816, 5,567,569, 5,561,029, 5,558,983,5,550,004, 4,491,629, 4,458,994 and many others). Suitable photoresistscan be selected by preparing coated substrates as described herein andassessing hydrophobic focusing of a hydrophilic liquid onto theresulting hydrophilic loci, such as by visual analysis of3-hydroxypicolinic acid (3-HPA) crystals. To make such assessment anaqueous formulation (14.5 nano liters) of 3-HPA is dispensed on andoverlapping the loci on the substrate. As the aqueous solvent evaporatesit leaves 3-HPA crystals. Successful focusing of the hydrophilic liquidresults in a crystal that conforms to the shape of the hydrophilic loci.If the focusing is not successful the crystallization will occur at thesite of dispensing, consequently overlapping the loci.

Suitable photoresist compositions for use herein are coatable liquidscontaining at a diazo photoactive compound with a resin, such as anovolak (phenolic) base resin for increased viscosity, suspended in anorganic solvent. The diazonapthaquinone (DNQ) sensitized phenolic resin(known as novolak resin) are widely available for wafer photolithographyprocesses. Such photoresist compositions are well known (see, e.g., U.S.patents cited above; such resins are commercially available from, forexample, Shipley Co., Marlboro, Mass. and Clarian Corp., Charlotte, N.C.and others) and any may be employed in the methods herein to produce thesubstrates provided herein. The most suitable are those that yield thebest hydrophobic focusing as described above. For example, AZ111XFSavailable from Clariant Corp., Charlotte, N.C., contains cresol novolakresin (117520-84-0), 2,1,4-diazonaphthoquinone ester with cumyl phenol,polyvinyl methyl ether, styrene/acrylic polymer in propylene glycolmonomethyl ether acetate.

Exemplary Substrate

FIG. 10 is a plan view of a microarray substrate 1000 for use in theFIG. 1 sample delivery system. An exemplary substrate 1000 can beconstructed using photolithographic techniques and hydrophobic materialsas described herein to form the target locations (loci) at which, forexample, material will be applied and at which liquid samples will bedeposited. The target locations on the microarray are defined with theapplication of photoresist materials and photolithographic depositionsuch that the target locations on the chip are less hydrophobic than thesurrounding areas. This differential hydrophobicity reduces theoccurrence of satellite droplets that might otherwise extend from theliquid sample and adhere to the microarray surface and permitsdeposition of small sample amounts.

FIG. 10 shows a 12×8 grid of target locations that have been formed on a3068 mm×1960 mm surface. Other densities of target locations may beobtained, as desired. The starting surface may be any material that hasan available —OH or primary amine, including SiO₂ and other forms ofglass, plastic, and “TEFLON”-brand materials (Trademark, E. I. DuPontfor polytetrafluoroethylene), such as any other material to which thesamples, matrix, molecules or biological particles of interest do notadhere, and include any other such materials that are commonly used inthe electronics industry to passivate electronic components and circuitboards, and materials used as a coating for medical devices, especiallyimplants, catheters, probes and surfaces of needles. As noted, anelement is defined as less hydrophobic than another by the relative“wettability” of the element or contact angles, where the contact angleof an element is less than the surrounding surface. For example,exemplary microarrays with differential hydrophobicity provided hereinhave SiO₂ surfaces with contact angles of 50-55 degrees and containarrays of target photoresist elements having contact angles of 65degrees. To create a less hydrophobic environment at each target site,the starting surface was treated with a 3.5% solution ofDiMethylDiChloroSilane (DMDCS) from United Chemical Technology ofBristol, Pa., USA in 95% Hexanes from EM Industries, Inc. of Hawthorne,N.Y., USA. The DMDCS does not stick to photoresist and provides asurrounding environment on the substrate whose contact angle is 90degrees.

With the techniques exemplified below, a target location on themicroarray is formed such that the outer area surrounding the targetlocation has a greater hydrophobicity than the inner target area. Thisincreases the accuracy of liquid sample dispensing by reducing orsubstantially eliminating any satellite droplets from the liquid samplethat might adhere to the outer area.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1

A flat substrate containing an array of less hydrophobic elementssurrounded by more hydrophobic elements, was prepared with an array ofphotoresist elements. To prepare the array, silicon dioxide (SiO₂) wasgrown on silicon wafers to a height of 3025 angstroms, ±5%.Alternatively, the SiO₂ can be grown to a height of about 1050angstroms. This process is performed by a “wet oxidation” method inwhich H₂ and O₂ gases are used in converting the Si to SiO₂.

A photoresist material (such as “AZ 111 XFS” photoresist from ClariantCorporation of Charlotte, N.C., USA) was spun onto the SiO₂ to athickness of 0.2 μm to 1.22 μm, with a height of about 1.0 μm. Thephotoresist was solidified by baking at 65 degrees Celsius for two tothree minutes. The surface was then exposed to light of 365 nmwavelength through a mask that blocked light at the target locations.The photoresist that was exposed to light in the unmasked portions ofthe substrate was then washed off with a phosphoric acid-baseddeveloper, leaving an array of photoresist pads having dimensions ofapproximately 200 μm²×1.0 μm. The wafer was then baked at 195° C. for 60minutes to remove any remaining solvents. The substrate was then silatedwith DMDCS. The microarrays can contain any desired number of loci, andtypically have 96-, 384-, 1536-loci. Higher densities of loci anddensities that are multiples of other than 96 also are contemplated.

In this example, the surface is silicon; any surface that has anavailable reactive group, such as —OH or a primary amine, including butnot limited to TEFLON® (polytetrafluoroethylene (PFTE)), glass,derivatized glass, plastics and other such materials may be used.

Example 2

In another process provided herein, a microarray was produced with aflat starting substrate having an array of SiO₂ elements surrounded by asilane surface, thereby creating an array of elements less hydrophobicthan the surrounding area. The resulting substrate has target locationsthat are bare silicon dioxide, and the surrounding regions are silatedwith DMDCS.

Silicon dioxide was grown on silicon wafers to a height of 3025Angstroms ±5%. Alternatively, the SiO₂ can be grown to a height of about1050 angstroms. This process is performed by a “wet oxidation” method inwhich H₂ and O₂ gases are used in converting the Si to SiO₂.

The resulting substrate was patterned with “MEGAPOSIT” SPR 900-0.8photoresist from Shipley Company, L.L.C. of Marlborough, Mass., USA inthe manner described above in Example 1. The wafer was then baked at 70°C. for 30 minutes to remove any remaining solvents. The patternedsubstrate was silated with 3.5% DMDCS for twenty minutes, as describedabove for Example 1. The photoresist pads were then removed by washingthe substrate in acetone for eight minutes at room temperature, therebydissolving the photoresist and exposing the SiO₂. The contact angles ofthe two materials DMDCS and the bare SiO₂ creates a less hydrophobicenvironment at each target location.

Example 3

In another process, a microarray was produced using a flat substratehaving an array of SiO₂ elements surrounded by a TEFLON®(polytetrafluoroethylene (PFTE)) surface, to create an array of targetelements less hydrophobic than the surrounding area.

Silicon dioxide was grown on silicon wafers to a height of 3025Angstroms ±5%. Alternatively, the SiO₂ can be grown to a height of about1050 angstroms. This process is performed by a “wet oxidation” method inwhich H₂ and O₂ gases are used in converting the Si to SiO₂.

The resulting substrate was patterned with “MEGAPOSIT” SPR 900-0.8photoresist from Shipley Company, L.L.C. of Marlborough, Mass., USA asdescribed above for Example 1. The resulting substrate was baked as inExample 2.

The patterned substrate was coated with a TEFLON®(polytetrafluoroethylene (PFTE)) coating, such as “PerFluoroCoat” fromCytonix Company of Beltsville, Md., USA, to a height of 148 to 1200Angstroms. The photoresist pads were removed by washing the substrate inacetone for eight minutes at room temperature, thereby dissolving thephotoresist and exposing the SiO₂. The contact angles of the twomaterials TEFLON® and SiO₂ create microarray target locations with aless hydrophobic environment than the surrounding area. For themicroarray of Example 3, the TEFLON® (polytetrafluoroethylene (PFTE))material has a contact angle of 110 degrees, and the SiO₂ material has acontact angle of 55 degrees.

In these examples, the areas of differential hydrophobicity on theproduced microarray reduces the occurrence of satellite droplets thatmight otherwise extend from the liquid sample and adhere to themicroarray surface, thereby increasing the volume dispensing accuracy.

The methods and apparatus provided herein have been described above interms of presently preferred embodiments so that an understanding of thepresent invention can be conveyed. There are, however, manyconfigurations for sample delivery processes and systems notspecifically described herein, but with which the present methods andapparatus and disclosure herein is applicable. The present inventionshould therefore not be seen as limited to the particular embodimentsdescribed herein, but rather, it should be understood that the presentinvention has wide applicability with respect to sample deliveryprocesses and systems generally. All modifications, variations, orequivalent arrangements and implementations that are within the scope ofthe attached claims should therefore be considered within the scope ofthe invention.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1-60. (canceled)
 61. A substrate for use in mass spectrometric analyses,comprising: an array of target locations defined by application of aphotoresist material onto a silicon solid support or a silicon dioxidesolid support, application of hydrophobic material to areas surroundingthe target locations and removal of the photoresist material, whereinthe array of target locations are less hydrophobic than the surroundingareas.
 62. The substrate of claim 61, wherein the hydrophobic materialis silane.
 63. The substrate of claim 63, wherein the silane isdimethyldichlorosilane (DMDMS).
 64. The substrate of claim 61, whereinthe hydrophobic material is polytetrafluoroethylene or a derivativethereof.
 65. The substrate of claim 61, comprising a silicon dioxidesolid support.
 66. The substrate of claim 61, wherein the array consistsof 24 target locations.
 67. The substrate of claim 61, wherein the arrayconsists of 96 target locations or 384 target locations.
 68. Thesubstrate of claim 61, wherein the target locations have contact anglesof about 50-55 degrees and the areas surrounding the target locationshave contact angles of about 90 degrees.
 69. The substrate of claim 61,wherein the photoresist material is a positive type photoresist.
 70. Thesubstrate of claim 69, wherein the photoresist material contains adiazonaphthoquinone sensitizer and a phenolic acid.
 71. The substrate ofclaim 61, wherein the silicon solid support or the silicon dioxide solidsupport comprises a silicon wafer.
 72. A method of making a substratefor use in mass spectrometric analyses, comprising: applying photoresistmaterial onto a silicon solid support or a silicon dioxide solidsupport, whereby an array of target locations is defined by the locationof the photoresist material on the silicon solid support or a silicondioxide solid support; applying hydrophobic material to areassurrounding the target locations; and removing the photoresist materialfrom the target locations, thereby exposing the silicon solid support orthe silicon dioxide solid support at the target locations.
 73. Themethod of claim 72, whereby the application of hydrophobic material isby silation.
 74. The method of claim 73, wherein the hydrophobicmaterial is silane.
 75. The method of claim 74, wherein the silane isdimethyldichlorosilane (DMDMS).
 76. The method of claim 72, wherein thehydrophobic material is polytetrafluoroethylene or a derivative thereof.77. The method of claim 72, comprising a silicon dioxide solid support.78. The method of claim 72, wherein the array consists of 24 targetlocations.
 79. The method of claim 72, wherein the array consists of 96target locations or 384 target locations.
 80. The method of claim 72,wherein the target locations have contact angles of about 50-55 degreesand the areas surrounding the target locations have contact angles ofabout 90 degrees.
 81. The method of claim 72, wherein the photoresistmaterial is a positive type photoresist.
 82. The method of claim 81,wherein the photoresist material contains a diazonaphthoquinonesensitizer and a phenolic acid.
 83. The method of claim 72, wherein thearray of target locations is produced by selectively removing from theareas surrounding the target locations the photoresist material appliedto the silicon solid support or the silicon dioxide solid support beforeapplying the hydrophobic material to the areas surrounding the targetlocations.